We will pursue aspects of the molecular biology of pE194, a 3500 base pair plasmid in Bacillus subtilis which confers resistance inducibly to the macrolide-lincosamide-streptogramin B antibiotics. The resistance is conferred via a specific N-methylation of adenine in 23S ribosomal RNA. We will continue studies on the post-transcriptional regulation of synthesis of a protein required for this resistance. Regulation is accompanied by adjustments in the stability of the mRNA for this protein. We will purify and characterize an rRNA methylase specified by pE194. We will pursue studies on gene-overlap in the pE194 genome. These studies include DNA, protein and RNA sequencing and the study of plasmid mutants. Finally, we will continue to characterize DNA replication control in pE194 focussing on defining and studying those regions of the genome which are required for replication and to produce a plasmid-specific trans-acting replication substance. We hope to eventually sequence the entire plasmid genome.